Conventional biarsenical probes (probes containing two arsenic atoms) such as FlAsH and ReAsH, which bind a tetracysteine tag incorporated into a protein of interest are utilized as affinity probes. These biarsenical probes have distinct advantages over other multiuse affinity probe systems such as the halo-tag, the SNAP tag, and fluorescent proteins in that the engineered peptide tag is small compared to entire protein domains. This allows utilization in many situations in which a larger tag prevents correct transport of the tag protein or biophysical measurements become inaccurate due to artificial distances.
Biarsenical probes have been utilized in imaging, biophysical fluorescent studies, fluorophore-assisted light inactivation, and for isolation of proteins and protein complexes. In addition to the original probes, FlAsH and ReAsH, improved fluorophores having improved properties have recently been synthesized. However, to date, these probes have equivalent distances between the two arsenic atoms (about 6 Å) and therefore have optimal binding to the same tetracysteine arsenic binding motifs (CysCysProGlyCysCys; SEQ ID NO. 3). Accordingly, the two color experiments utilizing such equidistance probes are limited to pulse-chase experiments on a single protein. Researches seeking to study proteins by fluorescence resonance energy transfer (FRET) have been limited to homo-FRET or use of fluorescent proteins as one of the FRET partners.
It would be advantageous to develop alternative biarsenical probes and probe binding peptide motifs.